Isolation and characterization of eight polymorphic microsatellite loci for Natterer's bat, Myotis nattereri (Vespertilionidae, Chiroptera)

Eight new microsatellite loci were isolated and characterized for the Natterer's bat Myotis nattereri from a microsatellite-enriched genomic library. The usefulness of these markers was assessed by screening a sample comprising 100 specimens collected from throughout the species range in Europe. Both moderately and highly polymorphic loci were identified with 3-17 alleles segregating per locus (mean 8.1 SE +/- A 0.048). No evidence for departure from HWE or linkage disequilibrium among loci was observed. These markers will provide a valuable addition to the molecular toolbox currently available for studies of population genetic structure, parentage and social organisation of M. nattereri and related species.

Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach

The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.

Genetic assessment of parentage in the caridean rock shrimp Rhynchocinetes typus based on microsatellite markers

Over the past decade, the common rock shrimp, Rhynchocinetes typus H. Milne Edwards, 1837, has been the focus of extensive investigations on mating behaviour. The species is now perceived as a model system for the study of reproductive strategies and sexual conflict in crustaceans displaying external fertilization. Using molecular markers, the current study assesses whether social mating behaviour in common rock shrimp translates into true genetic parentage. In a large mesocosm tank with >200 individuals of both sexes, the analysis of 15 families (22 eggs per female) for three informative microsatellites unambiguously confirmed multiple paternity in 11 instances (73%) involving, in each case, two to four males. Where more than one male was identified siring a particular brood, reproductive skew was apparent towards a single individual. Results suggest that multiple paternity in this species results from subordinate male coercive behaviour, female solicitation of multiple male matings or a combination of both.

Molecular pedigree reconstruction and estimation of evolutionary parameters in a wild Atlantic salmon river system with incomplete sampling: a power analysis

Background: Pedigree reconstruction using genetic analysis provides a useful means to estimate fundamental population biology parameters relating to population demography, trait heritability and individual fitness when combined with other sources of data. However, there remain limitations to pedigree reconstruction in wild populations, particularly in systems where parent-offspring relationships cannot be directly observed, there is incomplete sampling of individuals, or molecular parentage inference relies on low quality DNA from archived material. While much can still be inferred from incomplete or sparse pedigrees, it is crucial to evaluate the quality and power of available genetic information a priori to testing specific biological hypotheses. Here, we used microsatellite markers to reconstruct a multi-generation pedigree of wild Atlantic salmon (Salmo salar L.) using archived scale samples collected with a total trapping system within a river over a 10 year period. Using a simulation-based approach, we determined the optimal microsatellite marker number for accurate parentage assignment, and evaluated the power of the resulting partial pedigree to investigate important evolutionary and quantitative genetic characteristics of salmon in the system.

Results: We show that at least 20 microsatellites (ave. 12 alleles/locus) are required to maximise parentage assignment and to improve the power to estimate reproductive success and heritability in this study system. We also show that 1.5 fold differences can be detected between groups simulated to have differing reproductive success, and that it is possible to detect moderate heritability values for continuous traits (h(2) similar to 0.40) with more than 80% power when using 28 moderately to highly polymorphic markers.

Conclusion: The methodologies and work flow described provide a robust approach for evaluating archived samples for pedigree-based research, even where only a proportion of the total population is sampled. The results demonstrate the feasibility of pedigree-based studies to address challenging ecological and evolutionary questions in free-living populations, where genealogies can be traced only using molecular tools, and that significant increases in pedigree assignment power can be achieved by using higher numbers of markers.

Lack of genetic structure and evidence for long-distance dispersal in ash (Fraxinus excelsior) populations under threat from an emergent fungal pathogen: implications for restorative planting

Genetic analysis on populations of European ash (Fraxinus excelsior) throughout Ireland was carried out to determine the levels and patterns of genetic diversity in naturally seeded trees in ash woodlands and hedgerows, with the aim of informing conservation and replanting strategies in the face of potential loss of trees as a result of ash dieback. Samples from 33 sites across Northern Ireland and three sites in the Republic of Ireland were genotyped for eight nuclear and ten chloroplast microsatellites. Levels of diversity were high (mean A R = 10.53; mean H O = 0.709; mean H E = 0.765) and were similar to those in Great Britain and continental Europe, whilst levels of population genetic differentiation based on nuclear microsatellites were extremely low (Φ ST = 0.0131). Levels of inbreeding (mean F IS = 0.067) were significantly lower than those reported for populations from Great Britain. Fine-scale analysis of seed dispersal indicated potential for dispersal over hundreds of metres. Our results suggest that ash woodlands across Ireland could be treated as a single management unit, and thus native material from anywhere in Ireland could be used as a source for replanting. In addition, high potential for dispersal has implications for recolonization processes post-ash dieback (Chalara fraxinea) infection, and could aid in our assessment of the capacity of ash to shift its range in response to global climate change.

Population genetic analyses reveal distinct geographical blooms of the jellyfish Rhizostoma octopus (Scyhpozoa)

Understanding the spatial integrity and connectivity of jellyfish blooms is important for ecologists and coastal stakeholders alike. Previous studies have shown that the distribution of jellyfish blooms can display a marked consistency in space and time, suggesting that such patterns cannot be attributed to passive processes alone. In the present study, we used a combination of microsatellite markers and mitochondrial cytochrome oxidase I sequences to investigate genetic structuring of the scyphozoan jellyfish Rhizostoma octopus in the Irish and Celtic Seas. The mitochondrial data indicated far higher levels of population differentiation than the microsatellites: ΦST[MT] = 0.300 vs. ΦST[NUC] = 0.013. Simulation studies indicated that the low levels of nuclear differentiation were not the result of limited power because of low levels of polymorphism. These findings, supported by palaeodistribution modelling and mismatch distribution analysis, are consistent with expansion of R. octopus from a single, limited refugium after the Last Glacial Maximum, followed by subsequent isolation, and that the discrepancy between the mitochondrial and nuclear markers is a result of the nuclear loci taking longer to reach mutation–drift equilibrium following the expansion as a result of their four-fold larger effective population size. The populations studied are probably not well connected via gene flow, and thus genetically as well as geographically distinct, although our findings also highlight the need to use a combination of organellar and nuclear markers to enable a more complete understanding of population demography and structure, particularly for species with large effective population sizes.

High-resolution genetic analysis reveals extensive gene flow within the jellyfish Pelagia noctiluca (Scyphozoa) in the North Atlantic and Mediterranean Sea

Despite the importance of gelatinous zooplankton as components of marine ecosystems, both ecologically and socio-economically, relatively little is known about population persistence or connectivity in jellyfish. In the present study, we employed a combination of nuclear microsatellite markers and sequence data from the mitochondrial cytochrome oxidase I (COI) gene to determine levels and patterns of population genetic structuring in the holoplanktonic jellyfish Pelagia noctiluca across the northeast Atlantic Ocean and Mediterranean Sea. Our results indicate a high degree of connectivity in P. noctiluca, with little evidence of geographical structuring of genetic variation. A small but significant differentiation of Atlantic Ocean and Mediterranean stocks was detected based on the microsatellite data, but no evidence of differentiation was observed with the mtDNA, probably due to the higher power of the microsatellites to detect low levels of genetic structuring. Two clearly distinct groups of genotypes were observed within the mtDNA COI, which probably diverged in the early Pleistocene, but with no evidence of geographical structuring. Palaeodistribution modelling of P. noctiluca at the Last Glacial Maximum (LGM; ca. 21 KYA) indicated large areas of suitable habitat south of the species’ current-day distribution, with little reduction in area. The congruent evidence for minimal genetic differentiation from the nuclear microsatellites and the mtDNA, coupled with the results of the palaeodistribution modelling, supports the idea of long-term population stability and connectivity, thus providing key insights into the population dynamics and demography of this important species

Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.

Donor leukemia following allogeneic bone marrow transplantation

Although allogeneic bone marrow transplantation has been shown to be a highly effective treatment for acute and chronic leukemia, leukemic relapse remains a significant problem. Leukemic relapse occurs in recipient cells in the majority of cases, but the paucity of donor cell leukemias may reflect the sensitivity of the investigative technique. We have developed a highly sensitive technique to identify the origin of all hematopoietic cells in the post transplant state which is based on PCR amplification of microsatellites, polymorphic tandem repetitive elements. We have identified donor leukemia (AML M5) following a sex matched BMT for severe aplastic anemia, verified a previously reported case of donor leukemia following BMT for chronic granulocytic leukemia and recently identified an acquired cytogenetic abnormality(del 11q23) in donor cells four years following an apparently successful BMT for AML. In all cases the donors have remained healthy. Postulated mechanisms include transfer to the transplanted marrow of a dormant oncogene residing in the DNA of either a virus, the chromosomes of degenerating irradiation damaged host leukemic cells or in the marrow stroma which is radioresistant and host in origin following BMT. Using sensitive techniques donor leukemia has been shown to be a more common event than was previously thought and an understanding of its pathogenesis may allow us to elucidate leukemogenic mechanisms in man.

Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia:documentation of chimaerism using the polymerase chain reaction

A 3-year old child with juvenile chronic myeloid leukaemia received a T cell-depleted BMT from a male unrelated donor. There was early graft failure associated with increasing splenomegaly and hypersplenism. Splenectomy was performed 53 days post-transplant and was followed by autologous marrow recovery with return of leukaemia. A second unrelated donor BMT was performed 9 months later using T cell-replete marrow from a similarly matched female donor. Grade 2 GVHD involving the skin and gut responded to treatment with steroids. Chimaerism was assessed using Y-specific polymerase chain reaction (PCR) and microsatellites. Samples taken at the time of splenectomy showed no donor marrow engraftment but there was significant engraftment in the spleen. Following the second transplant, donor-type haematopoiesis was documented using a panel of microsatellite probes. The patient remains well 6 months after transplant. Splenectomy should be considered prior to transplant in patients with significant splenomegaly and hypersplenism. Partial chimaerism in the spleen, but not bone marrow, post-BMT, has not previously been documented. PCR technology is a useful and highly sensitive way to assess chimaerism post-BMT and is informative in sex-matched cases, whilst the small amount of material required is advantageous in paediatric patients.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Novel polymorphic microsatellite loci for the protogynous hermaphrodite slinger sea bream (Chrysoblephus puniceus, Sparidae)

As a result of the global decline of fish stocks, an increasing number of fish species are becoming targets of heavy exploitation, often concomitantly with a lack of biological knowledge on their structure and demographics. Here we present 11 new polymorphic microsatellite loci, isolated from the slinger sea bream (Chrysoblephus puniceus, Sparidae), a relatively recent target of coastal fisheries in eastern South Africa. Levels of genetic diversity were assessed in 39 individuals collected from the KwaZulu-Natal coast (Park Rynie, South Africa). Observed and expected heterozygosities varied between 0.39 and 0.97 and between 0.53 and 0.96, respectively. One locus (SL35) showed significant heterozygote deficiency and linkage disequilibrium was detected between SL35 and SL1. Importantly, five of these microsatellites cross-amplify in Cheimerius nufar, a sympatric species also subjected to exploitation.

Highly polymorphic microsatellite loci in the bank vole (Clethrionomys glareolus)

We describe seven polymorphic, dinucleotide microsatellite loci isolated from bank voles (Clethrionomys glareolus, Rodentia: Muridae) collected from the Wirral Peninsula, United Kingdom. Microsatellites were isolated as part of a long-term study on the wider effects of host-pathogen interactions of an endemic viral disease. These microsatellites showed between five and 13 alleles per locus in these populations. Observed and expected heterozygosities varied between 0.275 to 0.777 and 0.487 to 0.794, respectively. These markers will allow us to investigate the structure of this bank vole population. © 2005 Blackwell Publishing Ltd.

A primer for use of genetic tools in selecting and testing the suitability of set-aside sites protected from deep-sea seafloor massive sulfide mining activities

Seafloor massive sulfide (SMS) mining will likely occur at hydrothermal systems in the near future. Alongside their mineral wealth, SMS deposits also have considerable biological value. Active SMS deposits host endemic hydrothermal vent communities, whilst inactive deposits support communities of deep water corals and other suspension feeders. Mining activities are expected to remove all large organisms and suitable habitat in the immediate area, making vent endemic organisms particularly at risk from habitat loss and localised extinction. As part of environmental management strategies designed to mitigate the effects of mining, areas of seabed need to be protected to preserve biodiversity that is lost at the mine site and to preserve communities that support connectivity among populations of vent animals in the surrounding region. These "set-aside" areas need to be biologically similar to the mine site and be suitably connected, mostly by transport of larvae, to neighbouring sites to ensure exchange of genetic material among remaining populations. Establishing suitable set-asides can be a formidable task for environmental managers, however the application of genetic approaches can aid set-aside identification, suitability assessment and monitoring. There are many genetic tools available, including analysis of mitochondrial DNA (mtDNA) sequences (e.g. COI or other suitable mtDNA genes) and appropriate nuclear DNA markers (e.g. microsatellites, single nucleotide polymorphisms), environmental DNA (eDNA) techniques and microbial metagenomics. When used in concert with traditional biological survey techniques, these tools can help to identify species, assess the genetic connectivity among populations and assess the diversity of communities. How these techniques can be applied to set-aside decision making is discussed and recommendations are made for the genetic characteristics of set-aside sites. A checklist for environmental regulators forms a guide to aid decision making on the suitability of set-aside design and assessment using genetic tools. This non-technical primer document represents the views of participants in the VentBase 2014 workshop.